Clinical, Molecular, and Histopathological Correlates in a Series of 24 Consecutive Cases of Suspected Myeloproliferative Neoplasms with a JAK2V617F Variant Allele Frequency ≤1%

Background: JAK2^V617F is detected in >95% of polycythemia vera (PV) and 50-60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2^V617F variant allele frequency (VAF) is associated with different clinical characteristics and outcome. Highly sensitive molecular assays led to detect JAK2^V617F mutation also in healthy subjects, associated with a higher risk of coronary heart disease (N Engl J Med. 2017;377(2):111-121) in the context of CHIP. To date, comprehensive data on suspected MPN patients with a JAK2^V617F VAF≤1% are lacking.

Aim and methods: The aim was to analyze clinical, molecular, and histopathological correlates in a series of suspected MPN patients (pts) with a JAK2^V617F VAF≤1% (from 2017 to 2022) at our Center. JAK2^V617F VAF was measured in granulocytes by highly sensitive RTQ-PCR. Conventional karyotype and target next-generation sequencing (NGS) analysis were performed (complete data will be presented at the meeting). Bone marrow (BM) biopsies were reviewed by two independent expert pathologists, R.S. and U.G., the latter included as an author in the International Consensus Classification (Blood 2022; 2022015850).

Results: 21 males and 3 females (M/F=7:1), with a median age of 58 y (range, 22-81) were included. Median JAK2^V617F VAF was 0.2% (0.1-1). In the suspicion of MPN, complete diagnostic work-up was carried out in pts presenting with erythrocytosis (n=15), thrombocytosis (n=4), history of atypical thrombosis (n=3), splenomegaly (n=1) and leukocytosis (n=1), (Fig.1). In pts with erythrocytosis, median (range) Hb and Hct values were 17 g/dL (15.8-18.5) and 51.5% (50.4-54.7), whereas leukocytes (6.6 x 10⁹/L, 5-11) and platelet (258 x 10⁹/L, 172-440) were within normal range. EPO level (10 mUI/mL; 1.8-12.5) was subnormal in 3 pts. Abnormal karyotype was documented in one case (45,X,-Y[7]/46,XY[13]). Additional mutations were documented in 3 pts: HBB T124N, SH2B3 S186I and cKIT D816V. In pts with thrombocytosis, platelet range was 460-1450 x 10⁹/L; Hb, Hct and leucocytes values were normal. MPL W515K and CALR L367Tfs*46 additional mutations were in one and two pts, respectively. Considering pts with atypical thrombosis, one had a history of arterial events while the others experienced cerebral venous sinus and portal vein thrombosis. Thrombophilia screenings were negative. Karyotype was normal in two pts, whereas one had 45,X,-Y[3]/46,XY[20]. Additional CBL p.D460dup was detected in one case. The subjects investigated for splenomegaly/leukocytosis had normal karyotype, no additional mutations.

Morphological features consistent with PV were documented in 4 pts (Fig. 2, A-B). In one of these cases, multifocal dense infiltrates of CD117+/CD25+ mast cells were detected, fulfilling the diagnostic criteria for systemic mastocytosis with associated hematological neoplasm. In the other cases, microscopic examination showed age-adjusted normal or focally increased cellularity, regular or mildly expanded erythroid lineage with no/only minor increase in granulocytic lineage and a higher number of megakaryocytes with PV-like features (i.e. polymorphic forms without atypia and no dense cluster formation) (Fig. 1, C-D). Neither reticulin fibrosis nor excess blasts were detected and these pts had a final diagnosis of MPN unclassifiable (MPN-U), with a PV-like morphology. In pts with thrombocytosis histology was consistent with ET in three cases and overt-PMF in one. Among ET, one pts harbored MPLW515K with grade 1 reticulin fibrosis (Fig. 2, E-F), whereas the overt PMF had CALR type 1 additional mutation. BM biopsies of pts presenting with atypical thrombosis, leukocytosis and mild splenomegaly showed non-specific MPN-like features (including focally increased cellularity, mild expansion of erythroid or granulocytic lineages, higher number of megakaryocytes with a TE-like or a PV-like morphology) and were diagnosed as MPN-U.

Conclusions: This study highlights the importance of using highly sensitive assays to detect JAK2^V617F, as well as the need to search for CALR and MPL mutations, particularly in pts with thrombocytosis. Moreover, in pts presenting with erythrocytosis and JAK2^V617F positivity with VAF≤1%, histopathological diagnostic clues to MPN may be subtle as in the early phase of the disease, emphasizing the need to integrate histopathology with clinical and genetic data in order to address patients to proper clinical management.

Loscocco:Novartis: Speakers Bureau. Mannelli:Novartis: Speakers Bureau; Blueprint: Speakers Bureau. Coltro:Novartis: Speakers Bureau. Guglielmelli:Novartis, Abbvie: Other: Other member of advisory board, speaker at meeting. Vannucchi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Blueprint: Membership on an entity's Board of Directors or advisory committees; Morphosys: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSk: Honoraria, Membership on an entity's Board of Directors or advisory committees.